Increase in insecticidal toxicity by fusion of the cry1Ac gene from Bacillus thuringiensis with the neurotoxin gene hwtx-I.
A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 x 2.0 microm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC(50) (after 48 h) value of 5.12 microg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensis cry gene and other foreign toxin genes and recombinant strains with high toxicity.
Xia
LiQiu
L
College of Life Sciences of Hunan Normal University, Key Laboratory of Microbial Molecular Biology, Changsha, 410081, Hunan Province, China. xialq@hunnu.edu.cn
Long
XiaoShan
X
Ding
XueZhi
X
Zhang
YouMing
Y
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000633:toxicity*; D004731:Endotoxins / Q000235:genetics / Q000633:toxicity*; D005822:Genetic Vectors; D006460:Hemolysin Proteins / Q000235:genetics / Q000633:toxicity*; D007814:Larva / Q000187:drug effects; D007915:Lepidoptera / Q000187:drug effects; D007928:Lethal Dose 50; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000633:toxicity*; D030162:Reptilian Proteins / Q000235:genetics / Q000633:toxicity*; D013111:Spider Venoms / Q000235:genetics / Q000633:toxicity*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011993:Recombinant Fusion Proteins; D030162:Reptilian Proteins; D013111:Spider Venoms; C083624:huwentoxin I; C030956:insecticidal crystal protein, Bacillus Thuringiensis